QuantiNova Multiplex RT-PCR Kit

For fast, reliable quantification of up to 5 RNA targets in a single tube by multiplex real-time RT-PCR

Sensitive detection of up to 5 targets in 1 tube

Reliable quantification of low- and high-abundance targets

Direct amplification from a single cell or up to 800 ng template

Visual pipetting control to minimize human errors

Two-phase hot-start procedure for room-temperature setup

Internal control RNA for verification of successful RT-PCR

The QuantiNova Multiplex RT-PCR Kit enables fast and reliable quantification of up to 5 RNA targets in a single tube by multiplex real-time RT-PCR. A combination of various integrated safety features removes variables and prevents artifacts, ensuring reliable gene expression profiling. Our Q-Bond technology and optimized master mix promote ultrafast multiplex real-time RT-PCR within 1 hour. The combination of a unique two-phase hot-start procedure with our multiplex PCR buffer system ensures highly sensitive qRT-PCR on any real-time cycler without the need for optimization, and enables automated reaction setup at room temperature. The kit also provides protocols for extracted RNA and direct amplification from cultured cells, even down to a single cell. The optional QuantiNova Internal Control RNA can be used to monitor successful reverse transcription and amplification, and the visual pipetting control can be used to monitor correct pipetting.

The QuantiNova Multiplex RT-PCR Kit is intended for molecular biology applications. This product is not intended for the diagnosis, prevention, or treatment of a disease.

Mechanism of fast cycling during annealing.

[A] The proprietary PCR buffer contains Q-Bond, a molecule that facilitates the reduction of the annealing step to 5 seconds. Q-Bond increases the affinity of Taq DNA polymerases for short single-stranded DNA fragments, reducing the time needed for successful primer annealing. [B] Without Q-Bond, the primer and polymerase bind sequentially to the template, increasing the duration of the annealing step.

At ambient temperature the HotStaRTScript is inhibited by the RT-Blocker and the QuantiNova DNA Polymerase is kept inactive by QuantiNova Antibody and QuantiNova Guard. At 50°C the RT is activated while the QuantiNova DNA polymerase remains inactive. At 95°C the RT enzyme is denatured and the DNA polymerase is activated.

Accurate reaction setup indicated by the built-in pipetting control.

The master mix contains an inert blue dye. Combined with QuantiNova Yellow Template Dilution Buffer, the resulting solution turns green, indicating that the reaction was set up correctly.

Performance

The specialized master mix supplied with the QuantiNova Multiplex RT-PCR Kit allows easy setup of multiplex reactions and ensures that results are comparable to singleplex RT-PCR. The highly concentrated 4x master mix accommodates up to 800 ng template input and offers outstanding sensitivity even with up to 5-plex reactions.

The kit can clearly distinguish between small differences in the amount of template and provide accurate quantification of targets of widely differing abundance. Additionally, a simple protocol for direct amplification from cultured cells is provided, enabling RNA analysis even from a single cell. The unique two-phase hot-start procedure (see figure Principle of the novel QuantiNova two-phase hot-start mechanism) ensures outstanding specificity and allows room-temperature reaction setup, which is conveniently suited for automated procedures. The specially developed PCR buffer contains the additive Q-Bond, which significantly reduces annealing and extension times, allowing multiplex qRT-PCR in about one hour. The addition of a visual pipetting control increases in-process safety by minimizing human errors (see figure Accurate reaction setup indicated by the built-in pipetting control). The optional Internal Control RNA, which can be co-detected with targets, removes variation caused by inhibitors or other factors, allowing accuracy and reproducibility to be monitored. Overall, our ultrafast and in-process controlled multiplex qRT-PCR workflow increases efficiency by generating more insight from limited sample material.

Principle

The QuantiNova Multiplex RT-PCR Kit delivers highly sensitive and rapid results over a wide dynamic range on both standard and fast cyclers without optimization. The specially developed PCR buffer contains the additive Q-Bond, which significantly reduces annealing and extension times (see figure Mechanism of fast cycling during annealing).
Amplifying reference and target genes in the same reaction increases reliability by minimizing well-to-well variations and handling errors. Additionally, an Internal Control RNA is provided and can be simultaneously detected to monitor successful reverse transcription and qPCR.

The QuantiNova Multiplex PCR Buffer includes a balanced combination of K+ and NH4+ ions as well as the unique synthetic Factor MP. Together they promote stable and efficient annealing of primers and probes to the nucleic acid template, enabling high PCR efficiency (see figure Unique multiplex PCR buffer). The QuantiNova Multiplex RT-PCR Kit also uses a two-phase hot-start procedure (see figure Principle of the novel QuantiNova two-phase hot-start mechanism). This includes heat-mediated activation of both the HotStaRT-Script reverse transcriptase at 50°C and the PCR polymerase at 95°C. At low temperatures, the HotStaRT-Script forms a complex with an RT-Blocker molecule, leading to inactivation. At 50°C, the complex dissociates and the active HotStaRT-Script enzyme is released. The QuantiNova DNA Polymerase is kept in an inactive state by the QuantiNova Antibody and a novel additive, QuantiNova Guard, that stabilizes the complex. This improves the stringency of the PCR hot-start procedure and prevents extension of non-specifically annealed primers and primer–dimers. Within 2 minutes of raising the temperature to 95°C, the QuantiNova Antibody and QuantiNova Guard are denatured and the QuantiNova DNA Polymerase is activated, enabling PCR amplification. This unique two-phase hot-start enables reaction setup to remain stable for up to 2 hours at room temperature, preventing the creation of artifacts and also facilitating automated reaction setup.

The master mix supplied with the QuantiNova Multiplex RT-PCR Kit contains an inert blue dye that does not interfere with the real-time PCR, but increases visibility in the tube or well. The QuantiNova Yellow Template Dilution Buffer contains an inert yellow dye. When the template nucleic acid (diluted with the QuantiNova Yellow Template Dilution Buffer) is added to the master mix, the color of the solution changes from blue to green (see figure Accurate reaction setup indicated by the built-in pipetting control), providing a visual indication of correct pipetting and reaction setup.

Procedure

The QuantiNova Multiplex RT-PCR Kit contains a ready-to-use 4x master mix, eliminating the need to optimize reaction and cycling conditions. Simply add the RT mix, up to 800 ng template RNA and primer-probe sets to the master mix and follow the protocol in the handbook to get fast and reliable results on any real-time cycler. ROX reference dye is also provided in a separate tube, so the concentration can be adjusted for your particular instrument.
The kit also offers a simple and fast protocol for direct amplification from cultured cells. The recommended input amount ranges from 2000 cells down to a single cell, e.g., separated by serial dilution or using the QIAscout instrument.
The QuantiNova Internal Control RNA (QN IC RNA) is an internal amplification control that can be optionally used to test for successful reverse transcription and amplification. It is detected as a 200 bp internal control in the yellow channel on the Rotor-Gene Q or in the VIC/HEX dye channel on other real-time PCR instruments when using the QuantiNova IC Probe Assay. Alternatively, it can be detected in the red channel on the Rotor-Gene Q or in the Cy5 dye channel on other real-time PCR instruments when using the QuantiNova IC Probe Assay Red 650.

Applications

The QuantiNova Multiplex RT-PCR Kit can be used for multiplex gene expression analysis of RNA targets on any real-time cycler. To fully benefit from multiplexing, we recommend using an instrument that provides up to 5-plex capacity, such as the Rotor-Gene Q.